Small molecule-based chemotherapy and radiation therapies are two important clinical modalities for treatment of cancer. These therapies are often plagued with non-specific toxicity and collateral damage to vital organs and nearby non-cancerous tissues even though they show high efficiency towards solid tumor-regression. The modalities seemingly cease to work during the relapsing of disease possibly because of incomplete cell killing or residual cells acquiring drug resistance leading to the evolvement of cancer stem cell (CSC)-like behavior. These drug resistant cells are differentiated into highly aggressive cancer cells and often metastasize shortly into vital organs leading to enhanced mortality.
Glucocorticoid receptor (GR) is a nuclear hormone receptor residing ubiquitously in almost all cells including most cancer cells because of its vital role in gluconeogenesis. This receptor in a ligand-bound state acts as a transcription factor in nucleus through binding to glucocorticoid response elements (GRE) in the promoter sequences of various GR responsive genes and regulates their transcription.
Dexamethasone (dex), a synthetic glucocorticoid (GR hormone-like molecule) exhibits antiproliferative effect on several tissues of different origin (Corroyer, S. E. et al. 1997, Endocrinology, 138, 3677-3685; Ramalingam, A. et al. 1997, Mol. Endocrinology, 11, 577-586; Rider, L. G. et al. 1996, J. Immunol., 157, 2374-2380; Goya, L. et al. 1993, Mol. Endocrinology, 7, 1121-1132; Wattenberg, L. W., and Estensen, R. D. 1996, Cancer Res., 56, 5132-5135; Greenberg A. K. et al. 2002, Am. J. Respir. Cell Mol. Biol., 27, 320-328) as well as regulates and controls metabolism, development, inflammation, cell growth, proliferation and differentiation (Yamamoto K. R. 1985, Annu. Rev. Genet. 19, 209-252; Cole, T. J. et al. 1995, Genes Dev., 9, 1608-1621; Rogatsky, I. et al. 1997, Mol. Cell Biol., 17, 3181-3193). GR-mediated glucocorticoid-signaling potentiates a possible hypoxia related pathway leading to inflammation. As an anti-inflammatory agent, dex inhibits hypoxia inducible factor (HIF-1), which has direct role in mediating angiogenesis through up-regulation of VEGF (Leonard, M. W. et al. 2005, J. Immunol., 174, 2250-2257). Hence, dexamethasone (dex) is a very important and inexpensive drug-like substitute used in various pathological conditions. As a gene carrier agent, dex-spermine conjugate was used to deliver genes to airway epithelial with concurrent reduction of inflammation (Gruneich J. A. 2004, Gene Ther, 11, 668-674).
The viral based gene delivery is extensively used for their phenomenally efficient process of delivering genes to wide variety of cells. However, a number of problems including host toxicity, immunogenic responses and non-specific genomic integration of transferred gene make viral delivery a risky option for delivering genes. In comparison, non-viral gene delivery is a much more robust and clinically safe option compared to viral counterparts.
Banerjee, R. et al. U.S. Pat. Nos. 6,346,516 B1; 6,333,433 B1; 6503945 and 6541649; Banerjee, R. et al. 1999, J. Med. Chem., 42, 4292-4299, Singh R S et al. 2002, Chem Eur J., 8, 900-909 disclose, DODEAC (N,N-dihydroxyethyl, N,N-dioctadecyl ammonium chloride) and its generic structures, which forms cationic liposome using co-lipid cholesterol in membrane filtered water. Mukherjee, A. and Banerjee, R. Indian Application No. 1936/DEL/2006 and PCT/IN2007/000367, EP-2061514-A2 and Mukherjee, A. et al. 2009, Mol Ther., 17, 623-631 illustrate that upon associating dex with ‘DODEAC: cholesterol’ to formulate DODEAC: cholesterol: dex (called DX hereafter), cancer cells can be efficiently and GR-specifically targeted for the delivery of reporter and anticancer genes. It has also been shown that DX simultaneously translocates genetic and lipid cargo inside the cancer cell-nuclei.
Recently, a potent anti-cancer estrogen-structure-based drug, ESC8 (17-α-[3-(N,N-dioctyl,N-methyl-amino)-propan-1-yl]-17β-estradiol) has been developed anticipating similar observation in another nuclear hormone receptor, estrogen receptor (ER) in breast cancer cells. ESC8 kills human breast cancer cells with high selectivity irrespective of its estrogen receptor (ER)-expression status (Reddy, B. S., and Banerjee, R. Indian patent Application No. 0278/DEL/2007 and PCT Application No.; PCT/IN-07/00615; Sinha, S. et al. 2011, Mol. Cancer Res., 9, 364-374; U.S. Pat. No. 8,012,952).
Furthermore, it is known that Neuropilin (NRP-1) is a membrane protein highly expressed in several cell types, including many cancer cells. Expression of NRP-1 in tumor correlates with advanced tumor stage and poor prognosis in some specific tumor types. MAPK (Gray, M. J., et al. 2005, Cancer Res., 65, 3664-3670; Wey, J. S., et al. 2005, Br. J. Cancer., 93, 233-241), PI3K/Akt (Hong, T. M., et al. 2007, Clin. Cancer Res., 13, 4759-4768; Wang, L., et al. 2003, J. Biol. Chem., 278, 48848-48860) and Rho/Rac (Cao, Y., et al. 2008, Cancer Res., 68, 8667-8672; Wang, L., et al. 2003, J. Biol. Chem., 278, 48848-48860) signaling were found to be regulated by NRP-1 and to control cell migration, invasion, and apoptosis.
The observation that cancer cells have a self-renewal mechanism similar to that of stem cells raised the concept of cancer stem cells (CSC). Only a few CSCs from tumor populations result in tumor in the animal model. Lapidot's observation in 1994 that leukemia can be reconstituted in SCID mice with a single leukemia-initiating cell that had an immature (i.e. stem-like) phenotype has raised speculation that cancers are driven by a CSC (Lapidot, T. et al. 1994, Nature 367, 645-648). CSCs provide a useful insight for research into the effective treatment of cancer particularly in advanced, aggressive and relapsing phenotype of cancer. The ANV-1 cell line, the breast CSC-like cell with mesenchymal characters, was raised by immunoediting breast tumors in a neu-transgenic (neu-tg) mouse. The cells went through an epithelial-to-mesenchymal transition (EMT) and acquired the breast CSC character (Santisben et al. 2009, Cancer Res., 69, 2887-2895). It is a good model for the investigation of NRP-1 in CSCs. It has been found that ANV-1 cells express fourfold more NRP-1 than their parental mouse mammary cells with epithelial character.
Recently, there is an upsurge in the development of potential therapeutics for the targeting and killing CSCs for the treatment of relapsing cancer. CSC has a prominent role in maintaining the relapsing and drug resistant phenotypes. Small molecule inhibitors are developed that are targeted mainly to developmental pathways such as sonic hedgehog, wnt, notch etc. which are perennially involved in the production of CSC, an analogue of normal stem cells (Lukaszewicz, A. I. et al. 2010, J. Med. Chem., 53, 3439-3453). Liposomal delivery of differentiation inducing molecules such as retinoic acid is recently used as a concept to retard the onset of CSC-induced cancer relapse (Li, R. J. et al. 2011, J. Controlled Rel., 149, 281-291). Immunoliposomes targeted to CSC surface markers are also used to target and treat CSC-induced cancer (Zhang, J. 2010, J. Controlled Rel, 18, 675-687).
Targeting and treating aggressive, drug resistant and relapsing condition of cancer, a trait followed by the cancer stem cells and their subclonal populations, is a challenging task. Efforts are underway to develop drug molecules targeting developmental pathways which are involved in the initiation of CSC.
Furthermore, there is no direct evidence of cationic lipid-based therapeutics against cancer stem cell (CSC). Cationic liposomes are used to deliver only genetic cargo to normal stem cells. The results are expected to be extrapolated for CSC as well, but no direct evidence is known. However, non-cationic liposomes are available for use against CSC. The main disadvantages of existing prior art for CSC are:    a) small molecular weight drugs that are designed to target CSC are mainly targeted to sonic hedgehog, wnt//β-catenin, Notch etc. developmental pathways which are potentially hazardous to target because it may pose non-specific collateral damages to normal developmental pathways;    b) Immunoliposomes uses antibody to target CSC membrane marker proteins and cannot logically differentiate between markers expressed on normal cells and CSC. These liposomes targeting CSC are mostly non-cationic in nature and hence gene carrying capacity is limited.
Therefore, keeping in view the hitherto prior art, a new synergistic composition has been developed for simultaneously delivering anti-cancer gene and small, hydrophobic (lipophilic) anticancer molecule to treat CSC-associated tumors and other aggressive tumors. The present invention is cationic lipid-based and it can carry dual cargo: drugs and genes simultaneously. Uniquely, it targets CSC via a non-developmental pathway i.e., through the cytoplasmic protein glucocorticoid receptor (GR) hence avoiding non specific collateral toxicity to normal developmental pathways and such a liposomal system is not known to target for the delivery of cargo to CSC. Targeting CSC or aggressive pancreatic cancer cells through this ubiquitous pathway makes the drug resistant cells more drug sensitive.